SUMMARY

Marine elasmobranchs retain relatively high levels of urea to
counterbalance the osmotic strength of seawater. Oviparous species, such as
the little skate Raja erinacea, release encapsulated embryos that
hatch after about 9 months on the seafloor. To study the ureosmotic capability
of skate embryos, we measured a variety of possible osmolytes and
ornithine–urea cycle (OUC) enzyme activities in little skate embryos,
and determined their physiological response to dilute seawater (75% SW)
exposure relative to controls (100% SW). The urea:trimethylamine oxide (TMAO)
+ other osmolytes ratio was 2.3–2.7:1. At the earliest stage of
development investigated (4 months), there were significant levels of the key
OUC enzyme, carbamoyl phosphate synthetase III, as well as ornithine
transcarbamoylase, arginase and glutamine synthetase, providing evidence for a
functional OUC. Embryos (4 and 8 months) survived and recovered from exposure
to 5 days of 75% SW. There was a significant increase in the rate of urea
excretion (five- to tenfold), no change in OUC enzyme activities, and
significant decreases in the tissue content of urea, TMAO and other osmolytes
in embryos exposed to 75% SW compared to 100% SW. Taken together, the data
indicate that little skate embryos synthesize and retain urea, as well as a
suite of other osmolytes, in order to regulate osmotic balance with the
external environment. Interestingly, these ureosmotic mechanisms are in place
as early as 4 months, around the time at which the egg capsule opens and the
embryo is in more direct contact with the external environment.

Introduction

Elasmobranchs mostly inhabit marine environments, except for the strictly
freshwater genus Potomatrygon, and are therefore faced with the
dehydrating force of salt water. Elasmobranch body fluids are actually
slightly hyperosmotic to seawater (for a review, see
Holmes and Donaldson, 1969),
the predominant osmolyte being urea, which reaches concentrations up to 680
mmol l–1 in tissues, depending on the species (e.g.
Browning, 1978). Urea is toxic
at these concentrations; protein side chains become more fluid and normal
protein folding patterns are interrupted (for a review, see
Yancey, 2001b). In order to
counteract this extreme uraemia, other organic osmolytes that have
protein-stabilizing properties (primarily methylamines) are present in
elasmobranch tissues. Trimethylamine oxide (TMAO) is generally the principle
counteracting osmolyte in elasmobranchs. The importance of other organic
stabilizers varies depending on species, but typically includes sarcosine
(Forster and Goldstein, 1976)
and betaine (Robertson, 1975;
Bedford et al., 1998). Taken
together, the various counteracting osmolytes have additive effects on urea
destabilization and have been found to perform optimally in vitro
when the ratio of urea to methylamines is 2:1 (for a review, see
Yancey, 2001a). This ratio is
well conserved across species, supporting the hypothesis that it is
physiologically important for optimum macromolecule stability.

There is very little information on the tissue organic osmolyte ratio in
early life stages. In the big skate Raja binoculata embryo, the ratio
of urea:TMAO can be calculated as approximately 3.4:1 (from data reported by
Read, 1968a). Possibly
embryonic elasmobranchs are unable to maintain the typical 2:1 ratio, or other
organic osmolytes that have not been measured play an important role, or
higher ratios are the norm for early life stages. Thus, fundamental
information is missing on the role of compatible and counteracting osmolytes
in early life stages.

As in mammals, urea in elasmobranchs is synthesized via the
hepatic ornithine urea cycle (OUC), which is composed of the enzymes carbamoyl
phosphate synthetase III (CPSase III), ornithine transcarbamoylase (OTCase),
argininosuccinate synthase (ASS), argininosuccinate lyase (ASL) and arginase.
Unlike the mammalian CPSase I that prefers ammonia as a nitrogen-donating
substrate, elasmobranch CPSase III requires glutamine, and therefore glutamine
synthetase (GSase) is a necessary accessory enzyme for this pathway. Is the
OUC functional in embryonic elasmobranchs? Read
(1968a) discovered that total
urea content increased in the oviparous R. binoculata with embryonic
mass (and presumably age) and that they maintain relatively constant
concentrations of urea and TMAO in both the embryonic body and yolk throughout
embryonic development. Evans and Kormanik
(1985) removed embryos
prematurely from the uterus of the viviparous spiny dogfish Squalus
acanthias and reported that they maintain physiological concentrations of
blood urea for at least 3 days. This very early ureosmotic capability may be
due to the presence of a functional OUC in embryonic elasmobranchs. Read
(1968b) did indeed establish
the presence of two OUC enzymes, OTCase and arginase, in the embryonic bodies
(but not the yolks) of the spiny dogfish S. suckleyi and R.
binoculata. CPSase III is considered the rate limiting enzyme of the OUC
(Anderson, 1995) and GSase is
an important auxillary enzyme that supplies the nitrogen donating substrate
glutamine. To our knowledge, these enzymes have never been measured in the
embryos of any species of elasmobranch. Thus, the co-expression of these four
enzymes (GSase, CPSase III, OTCase and arginase) would provide support for the
possibility of a functional OUC early in development. In the absence of CPSase
III, urea synthesis via arginolysis or uricolysis occurs
(Mommsen and Walsh, 1992).

Our understanding of the development of physiological regulations, such as
osmoregulation, in non-mammalian species has been described as `embryonic'
(Spicer and Gaston, 1999).
While previous studies have been conducted on mature individuals, dilute
seawater challenge has never been studied in embryonic elasmobranchs. Smith
(1936) proposed that the
embryos of elasmobranchs are afforded early protection from osmotic stress
either by encapsulation in a sealed egg case (in the case of oviparity) or by
incubation in osmotically stable intrauterine fluids (in the case of
viviparity). Embryos of oviparous species, however, are exposed directly to
seawater well before hatching (Wourms,
1977) and may be able to independently ureosmoregulate by altering
the rate of urea excretion and/or the rate of urea synthesis.

The first objective of this study was to determine the full suite of
organic osmolytes retained in embryonic elasmobranchs and the ratios of
urea:TMAO and other osmolytes. Secondly, we wanted to establish the presence
(or absence) of the key regulatory OUC enzyme, CPSase III, as well as related
OUC enzymes, in early life stages. Finally, we wanted to determine the ability
of elasmobranch embryos to adjust their ureosmotic parameters under dilute
water stress. We predict that if embryos have the capacity to acclimate to a
lower external salinity, they may increase the rate of urea excretion and/or
decrease the activities of OUC enzymes in order to downregulate tissue urea
levels. Captive little skates Raja erinacea at the Hagen Aqualab,
University of Guelph, Canada, release multiple egg cases each year. The
embryos develop for approximately 9 months before hatching (at 10°C). In
contrast to Read's studies (Read,
1968a,b)
where increasing age was assumed by increasing embryonic mass, the precise
dates of oviposition were recorded for individual embryos used in the present
experiment. To address our first objective, urea, TMAO and 12 other potential
osmolytes (glucose, myo-inositol, taurine, glycerophosphorycholine (GPC),
serine, betaine, glycine, proline, creatine, β-alanine, glutamine and
sarcosine) were measured in 4- and 8-month post-oviposition embryonic body and
yolk, and the ratio of urea to other osmolytes was calculated. The activity of
the OUC enzymes CPSase III, OTCase and arginase, as well as the accessory
enzyme GSase were measured at both stages. Finally, osmotic stress was induced
by exposing the embryos to 100% (control) or 75% seawater for 5 days, and the
embryos were subsequently recovered in 100% seawater for 3 days. Urea and
ammonia excretion, tissue osmolytes and urea cycle enzymes were measured in
both groups.

Materials and methods

Animals

Sexually mature little skates Raja erinacea Mitchill (mass∼
500 g) held in captivity at the Hagen Aqualab, University of Guelph,
Canada, regularly produced embryos, which they deposited onto a sand
substrate. Upon oviposition the embryos were removed from the adult tank,
tagged with the date of oviposition, and placed in separate (100 l, 1.2
m×0.6 m×0.2 m) tanks supplied with recirculating artificial
seawater (Crystal Sea, Baltimore, MA, USA; 33 p.p.t., pH 8.2, 10°C) for
the duration of their development (approximately 9 months). The embryos were
not fed during the experiment as they were feeding endogenously before
hatching. The embryo mass was approximately 0.35 g (at 4 months) and 4.13 g
(at 8 months).

Experimental protocol

Prior to exposure to dilute seawater (SW), embryos (4 and 8 months) were
removed from their egg cases by making a small incision at one end of the case
and gently tipping the animal into the experimental chamber. By removing the
egg case, nitrogen excretion rates to the external environment could be
measured directly rather than by sampling both inside and outside the egg case
in intact individuals. In preliminary experiments on 3–4-month embryos,
urea and TMAO embryonic tissue and yolk contents were measured in intact
embryos. There were no differences between these embryos and those in the
present study (N=4–6); for example, urea in the embryonic
tissue (382±26.2 mmol kg–1 tissue water) and yolk
(448±45.2 mmol kg–1 tissue water) of the intact
embryos was not significantly different from the values in decapsulated
4-month embryos presented in this study. Furthermore, Leonard et al.
(1999) compared metabolic rate
(i.e. oxygen consumption) in late term intact versus decapsulated
embryos and found no significant differences. Embryos were placed individually
into 250 ml plastic beakers with perforations, to allow for water renewal, and
were then placed in individual 12 l chambers of either 100% (33 p.p.t.,
control) or 75% (25 p.p.t., dilute) aerated SW (10°C). Dilute SW was made
by mixing seawater from the system with deionized water to a salinity of 25
p.p.t. (this dilution did not affect the pH or temperature of the water).
Water in all chambers was changed every 48 h to ensure that excreted ammonia
or urea was less than 20 μmol l–1.

Excretion rates were measured by placing the beaker in which the animal was
housed in a new, unperforated beaker containing either 60 ml (4-month) or 70
ml (8-month) of fresh 100% or 75% SW for a 3 h flux period. Water samples (15
ml) were collected at 0 and 3 h and stored at –20°C (for up to 6
weeks) for later analysis of ammonia and urea content. Urea and ammonia
excretion rates were calculated as previously described by Wright and Wood
(1985). This procedure for
measuring nitrogen excretion rates was repeated every 24 h up to and including
120 h after the onset of exposure to 100% or 75% SW. It should be mentioned
that preliminary trials using various volumes of water and flux periods were
conducted, and the volumes and times chosen were those that produced the best
representation of excreted ammonia and urea at both developmental stages.
After the final flux period, embryos were removed from their chambers, briefly
blotted dry and weighed. In the case of 4-month embryos, the embryonic body
was carefully dissected from the yolk sac. The embryonic body was then quickly
weighed, placed in a cryovial and flash frozen in liquid nitrogen. For 8-month
animals, whole embryos were first weighed and then killed by cervical section.
Yolk was quickly sampled from both 4- and 8-month stages (approximately
0.5–1 ml) by draining directly into a cryovial and frozen. Excess yolk
was removed from the 8-month body cavity by wiping with a Kimwipe until no
yolk remained. Animals were then weighed again and flash frozen in liquid
nitrogen.

Water content (%) of embryos and yolk samples was determined in a separate
group of embryos exposed to 75% or 100% SW for 120 h as described above. After
wet mass had been recorded, all samples were placed in an oven (55°C) for
drying until a constant dry mass was achieved. Percent water was calculated as
wet mass (g) – dry mass (g) / wet mass (g) × 100.

In order to determine the time to recovery after exposure to 75% SW, a
separate group of 4- and 8-month embryos were exposed to dilute seawater for
120 h as described above and then recovered in 100% SW. Excretion rates were
measured as described above over a 3 h period immediately following
replacement in 100% seawater and every 24 h thereafter for 72 h. Tissues were
sampled at the end of the recovery period as described above.

Water sample analysis

Water samples were analyzed for urea content using a colorimetric assay
described by Rahmatullah and Boyde
(1980). The detection limits
of this assay were 0.1–100 μmol l–1 urea.

The ammonia concentration in water samples was measured using the
Indophenol Blue method described by Ivancic and Degobbis
(1984). The detection limits
of this assay were 0.1–300 μmol l–1 ammonia.

Tissue osmolyte analysis

Embryos were ground to a fine powder using a mortar and pestle under liquid
nitrogen. Urea was measured using the method described by Rahmatullah and
Boyde (1980). All tissue and
yolk samples were prepared for urea analysis as described by Steele et al.
(2001), with the following
exceptions. Tissue supernatant was diluted an additional 500-fold for a total
dilution of 5000× (in order to bring the concentrations within the
detection limits of the assay), and instead of 8% perchloric acid, 5%
trichloroacetic acid (TCA) was used for deproteinization (no difference in
urea content was observed when TCA was substituted; data not shown).

TMA/TMAO was measured in both embryonic tissue and yolk using the ferrous
sulphate/EDTA method described by Wekell and Barnett
(1991). Samples (ground embryo
or yolk) were dissolved in 10 volumes of 5% TCA (samples were vortexed
extensively to ensure homogeneous mixing) and centrifuged at 16 000
g for 10 min. The supernatant was diluted an additional
fivefold with 5% TCA for TMAO analysis. All spectrophotometric measurements
for urea, ammonia and TMA/TMAO were made using a Perkin Elmer UV/VIS Lambda 2
spectrophotometer (Perkin Elmer Corp., Norwalk, CT, USA). The detection limits
of this assay were 0.5–3 mmol l–1 TMA.

In order to account for the presence of any potential remaining endogenous
substrates, controls without exogenous substrate were included in each enzyme
assay. Enzyme reactions for GSase, CPSase III, OTCase and arginase were run
according to Steele et al.
(2001), except that GSase,
OTCase and arginase activities were determined by the amount of product
produced from 0 to 6 min, while the CPSase III reaction was stopped after 18
min. Trial experiments using ammonia (NH4Cl) as the N-donating
substrate showed that activity was less than 5% of total CPSase activity
occurring in the presence of glutamine (data not shown). Enzyme activities
were measured at 26°C.

Statistical analysis

Differences between osmolyte contents (TMAO, urea, total other osmolytes)
or enzyme activities in 100% and 75% SW groups were determined using a General
Linear Models means comparison procedure, and urea and ammonia excretion rates
were analyzed using repeated-measures analysis of variance (ANOVA) with a
General Linear Models procedure, both using the SAS system (version 8e; SAS
Institute Inc., Cary, NC, USA). A Tukey test was applied if significant
differences were detected between control and 75% seawater exposed animals.
Statistical significance in all tests was declared at P<0.05. Due
to the limited number of embryos available for these experiments, a very small
number of animals (N=3 or 4) was used in all experiments. Data are
presented as means ± s.e.m.

Results

Tissue osmolytes in 100% seawater: yolk vs. embryo

Water content of 4- and 8-month embryo and yolk samples was significantly
higher after 120 h of exposure to 75% SW relative to 100% SW controls
(Table 1). Tissue levels of
urea, TMAO and other osmolytes were therefore corrected for tissue water
content (Table 2). The sum of
eleven `other' osmolytes (–glucose) constitutes the total of osmolytes
besides urea and TMAO. Glucose is not normally considered an important organic
osmolyte and was only detected in the yolk
(Table 2). Several individual
organic osmolytes were significantly different between the embryonic tissues
and the yolk in 4- and 8-month embryos
(Table 2). Myo-inositiol and
taurine were the only two osmolytes that were significantly higher in
embryonic tissue relative to yolk at both the 4- and 8-month stage, whereas
serine, proline and TMAO were significantly lower in both stages.β
-alanine, betaine and sarcosine were significantly lower in 4-month
embryo versus yolk. β-alanine, sarcosine and creatine were
significantly higher in 8-month embryo versus yolk.

Content of various osmolytes in the embryo and yolk of 4- and 8-month
old Raja erinacea embryos exposed to 100% or 75% seawater for 5
days

Tissue osmolytes in 100% seawater: 4-month vs. 8-month

There were also a number of significant differences between the 4- and
8-month embryos. Contents of several osmolytes (betaine, creatine, sarcosine
and β-alanine, with the latter rising the most) increased (3- to 12-fold)
from 4 to 8 months, although taurine and glutamine decreased by 2.8- and
2.1-fold, respectively (Table
2). Interestingly, an almost opposite trend was observed in the
yolk where osmolyte levels (taurine, proline, β-alanine, sarcosine) were
significantly lower in the 8-month relative to the 4-month old yolks, except
for GPC, which increased over fivefold, and glycine, which increased to
detectable levels (Table
2).

The total of all the organic osmolytes measured (minus TMAO and urea), as
well as urea alone, was lower in 4-month versus 8-month embryonic
tissue (Table 2). Tissue ratios
of urea:TMAO and other osmolytes were significantly higher in 8-month
embryonic tissue and yolk compared to 4-month embryos
(Table 2).

Response to dilute (75%) seawater

No mortalities occurred in either 4- or 8-month embryos due to dilute
seawater exposure or recovery. Urea excretion rates were significantly
increased (tenfold) in 4-month embryos at the onset of exposure to 75% SW
(over the first 3 h), and although excretion fell by 24 h, rates remained
significantly elevated over the control (100% SW) rate
(Fig. 1). Excretion rates
returned to control levels by 48 h of 75% SW exposure and remained there for
the rest of the 120 h exposure period. Upon return to 100% seawater, urea
excretion was slightly, but significantly elevated over control rates for up
to 24 h but returned to control values by 48 h
(Fig. 1). Similarly, in 8-month
embryos, there was a significant increase in urea excretion (fivefold) just
after exposure to 75% SW, with excretion rates returning to control levels by
48 h (Fig. 2). Urea excretion
rates increased significantly (11-fold) at the onset of recovery in 100% SW
but returned to control levels 24 h into recovery
(Fig. 2). Ammonia excretion was
not detected in 4-month embryos; however, in 8-month embryos ammonia excretion
constituted approximately 20–66% of total nitrogen excretion [% ammonia
excretion = (ammonia N excretion rate / ammonia N excretion rate + urea N
excretion rate) × 100] over the course of the experimental period
(Fig. 3). There was no change
in ammonia excretion in response to dilute seawater exposure; however, ammonia
excretion significantly increased (1.4-fold) at the onset of 100% SW recovery
(Fig. 3).

Urea excretion rates (mean ±
s.e.m.) in 4-month old Raja erinacea
embryos over a 120 h period of exposure to 75% seawater (A) and a subsequent
78 h reintroduction to 100% seawater (B), N=4. *Significantly
different from control value at that time point (P<0.05).

Urea excretion rates (mean ±
s.e.m.) in 8-month old Raja erinacea
embryos over a 120 h period of exposure to 75% seawater (A) and a subsequent
78 h reintroduction to 100% seawater (B), N=4. *Significantly
different from control value at that time point (P<0.05).

Tissue TMAO content was significantly lower in 4-month embryos exposed to
75% SW for 120 h, with full recovery by 72 h in 100% seawater
(Table 2,
Fig. 4). Similarly, glycine was
significantly decreased in the embryonic tissue of 4-month embryos exposed to
75% SW (Table 2). However,
levels of other osmolytes in the embryo, and all osmolyte levels in the yolk,
did not change between 100% and 75% SW
(Table 2,
Fig. 4). Contents of tissue
urea and several other osmolytes were significantly lower in 8-month embryos
after 120 h of 75% SW exposure, with full recovery by 72 h
(Table 2,
Fig. 5). GPC and glycine
decreased significantly in 8-month embryonic tissue and yolk after 75% SW
exposure, and β-alanine was significantly decreased in the embryonic
tissue alone (Table 2). Many
other individual osmolytes in both tissues of both stages showed lower
averages after 75% SW exposure (Table
2), but were not statistically different.

Stacked plot of urea, TMAO, and total other osmolytes content (mean±
s.e.m.) in 4-month old embryos (E)
and yolk (Y) samples after both 120 h of 75% seawater exposure (D) and 78 h of
recovery in 100% (R) seawater, N=4. C, control. *Significantly
different from control value (P<0.05).

Stacked plot of urea, TMAO, and total other osmolytes content (mean±
s.e.m.) in 8-month old embryos (E)
and yolk (Y) samples after both 120 h of 75% seawater exposure (D) and 78 h of
recovery in 100% (R) seawater, N=4. C, control. *Significantly
different from control value (P<0.05).

CPSase III and other OUC enzyme activities (GSase, OTCase and arginase)
were detected in both 4- and 8-month embryos
(Table 3). There were no
significant differences between enzyme activities and developmental stage.
Additional enzyme assays were performed on skeletal muscle tissue isolated
from 8-month embryos (Gsase 1.26±0.42; CPSase III 0.008±0.001;
OTCase 10.02±1.97; arginase 1.22±0.17 μmol
min–1 g–1, N=6). Whole embryo
CPSase III and other OUC enzyme activities were unchanged by exposure to 75%
SW exposure, either on a per g wet mass or a per mg protein basis
(Table 3).

Discussion

Our study provides the first evidence that embryos of the little skate
R. erinacea have a full range of osmoregulatory strategies in place
well before hatching. Although the literature is sparse, previously published
data indicated that skate and dogfish embryos maintain elevated urea levels
independent of the mother (Read,
1968a,b;
Evans and Kormanik, 1985). Our
findings confirm these earlier reports and extend previous observations in
three ways.

First, by measuring the contents of the destabilizing osmolyte urea and an
array of compatible and counteracting osmolytes, we have established the
urea:TMAO + other osmolyte ratio to be 2.3–2.7:1, similar to published
values for adult elasmobranchs (2–2.4:1; reviewed by
Yancey and Somero, 1980). As
in adult elasmobranchs, these ratios may be closer to 2:1 in the intracellular
compartment, because osmolyte analyses were conducted on whole-tissue
extracts. Urea is generally equilibrated between intra- and extracellular
fluids (Forster and Goldstein,
1976), thus the whole-tissue urea content per kg water
(Table 2) should be fairly
close to actual in vivo concentrations (mmol l–1).
However, the other organic osmolytes are typically more concentrated in the
intracellular compared to extracellular compartment
(Forster and Goldstein, 1976).
Hence, the whole-tissue organic osmolyte content
(Table 2) is probably less than
actual in vivo intracellular concentrations
(Yancey and Somero, 1980).
Nevertheless, the ratios of urea:TMAO+other osmolytes were determined using
whole-tissue extracts in both adults
(Yancey and Somero, 1980) and
embryos (present study) and are therefore comparable.

Second, the presence of significant levels of the rate-limiting OUC enzyme
CPSase III, along with OTCase, arginase and GSase, strongly suggests that the
OUC is functional very early in development. In particular, the activity of
CPSase III is more than sufficient to account for urea excretion rates in
embryos (Table 3, Figs
1,
2; for example, see
Appendix).

Third, no mortalities occurred in embryos exposed to and recovered from a
5-day exposure to dilute seawater (75%), an osmotic shock that would drive
water in and osmolytes out of embryonic tissues. Furthermore, by studying
embryos of known age we were able to differentiate between the osmoregulatory
abilities of very young embryos (4 months, 0.35 g embryonic tissue only) from
more developed, but still encapsulated embryos (8 months, 4.13 g embryonic
tissue only). Although there were some marked differences between 4- and
8-month embryos (see below), overall we can conclude that little skate embryos
synthesize and retain urea, as well as a suite of other osmolytes, in order to
regulate osmotic balance with external seawater.

Developmental differences were observed in the osmolyte ratios and the
response to environmental dilution. The urea:TMAO + other osmolyte ratio was
significantly higher in 8-month than in 4-month embryos
(Table 1). This difference is
due, for the most part, to the significantly higher (+27%) urea levels in the
8-month old embryos, but only modestly higher levels of other osmolytes and
similar TMAO contents (Table
2). There were also differences in individual osmolytes other than
urea and TMAO. Taurine was the principle `other' osmolyte found in the
embryonic tissue of 4-month embryos, but was significantly lower in the
8-month stage. The predominant `other' osmolyte at 8 months was β-alanine
(Table 2). Sarcosine was also
significantly higher in 8-month than in 4-month embryonic tissue
(Table 2). As well as having
protein stabilizing properties (Yancey and Somero,
1979,
1980), these compounds are
arguably some of the most important free amino acids involved in cell volume
regulation in fish (King and Goldstein,
1983). Taurine and β-alanine in particular are distributed in
comparatively large quantities in a variety of tissues, such as the
elasmobranch brain, red blood cells, myocardium and muscle, while sarcosine is
a major component of the amino acid pool in skate muscle (for a review, see
King and Goldstein, 1983).
Their importance as osmolytes probably stems from the fact that, in addition
to being compatible or stabilizing towards proteins, they are relatively
metabolically inert compared to other amino acids and can therefore be
accumulated without interfering with metabolic pathways within the cell
(King and Goldstein, 1983). It
would be logical to consider, therefore, that early life stages may be
actively regulating the metabolism and retention of these amino acids as
development progresses to establish the high physiological levels required in
adulthood.

The relative balance between TMAO and other compatible and stabilizing
osmolytes varies between species and possibly between developmental stages.
Some shark tissues are known to have relatively high TMAO levels, whereas the
little skate appears to retain lower TMAO levels but higher levels of
osmolytes such as sarcosine, β-alanine, taurine, etc.
(King and Goldstein, 1983).
Our data indicate that TMAO levels are relatively high (two- to threefold
higher) at both embryonic stages (Table
2) compared to TMAO levels in adult muscle cells and plasma (64
mmol l–1 and 39 mmol l–1, respectively;
Forster and Goldstein, 1976)
and adult liver and muscle tissue (∼40-50 mmol kg–1
tissue water; S. Steele, P. Yancey, P. Wright, manuscript submitted for
publication). The total of other osmolytes in embryos are comparable to adult
liver tissue, but are only about one half of the total in adult muscle tissues
(∼180 mmol kg–1 tissue water, S. Steele, P. Yancey, P.
Wright, manuscript submitted for publication). The embryonic total osmolytes
are also low compared to published values for adult wing muscle taurine,
sarcosine, proline, glycine and β-alanine reported by Boyd et al.
(1977), which add up to
approximately 92 mmol kg–1. When considering the collection
of compatible and counteracting osmolytes in the little skate, therefore, the
present data indicate that TMAO may play a larger role than other non-urea
osmolytes in embryos than in adults.

Yolk levels of urea, TMAO and other osmolytes are in most cases
significantly higher compared to embryonic tissues when calculated as per
tissue water (Table 2). If one
calculates contents based on the wet mass of the tissue, however, yolk
osmolyte contents are typically lower than comparable embryonic tissue levels
(data not shown), due to the much lower water content
(Table 2). The yolk of teleost
species ranges from 54% water in freshwater salmonids (e.g.
Rombough, 1988) to 93% water
in marine pelagic teleosts (e.g. Ying and
Craik, 1993). As discussed above, in vivo tissue
concentrations may be the same (urea) or higher (other organic osmolytes) than
whole-tissue contents, but the situation may be more complex in yolk because
of its composition. The distribution of urea and other osmolytes in the yolk
water, yolk lipid and extracellular fluid is unknown, but is necessary in
order to understand the actual in vivo yolk concentrations.

TMAO content was significantly lower in 4-month embryos (–27%) but
not in 8-month embryos exposed to 75% seawater. In contrast, the total of
other osmolytes was significantly lower in the 8-month embryos (–33%)
but not in the 4-month embryos. Glycine was downregulated to undetectable
levels in both 4- and 8-month embryonic tissue, although it only constitutes
2–3% of the total value for other osmolytes and is not strongly
implicated in tissue osmotic regulation in adult elasmobranchs. Boyd et al.
(1977) reported a significant
decrease in erythrocyte glycine content as well as brain, muscle and
erythrocyte β-alanine levels in R. erinacea exposed to 50% SW.
Our findings agree with the adult data, in that there was a large decrease inβ
-alanine in 8-month embryonic tissue with exposure to 75% SW. Three of
the solutes measured here – myo-inositol, GPC and creatine – have
not been typically analyzed in elasmobranch osmotic studies, with a couple of
exceptions (see Yancey,
2001a). They are included here because they have been shown to act
as osmolytes in other animals and tissues, especially mammals
(Yancey, 2001b). Creatine did
not exhibit any significant changes between 100% and 75% SW; it most likely
has a different primary function, e.g. in energy storage with creatine
phosphate. GPC acts like a regulated osmolyte at the 8-month stage as it is
significantly lower in these embryos exposed to 75% seawater
(Table 2). Whether myo-inositol
can be considered to function as an osmolyte in this animal will require
further study.

Urea content in the embryonic tissue decreased significantly in the tissues
of 8-month (–20%), but not 4-month, embryos exposed to 75% SW
(Table 2). As predicted, urea
excretion rates were initially elevated in both the 4- and 8-month embryos
following the onset of 75% SW exposure. In fact, the magnitude of this
response was much higher in the 4-month embryos
(Fig. 1) relative to the
8-month embryos (Fig.
2).Indeed, the difference in whole embryo urea content (tissue
plus yolk) between control and 75% seawater exposed embryos is approximately
68 mmol kg–1 wet mass, less than that lost by excretion over
the entire 5 day period (145 mmol kg–1 wet mass). In 8-month
embryos, however, the decrease in urea content of the whole embryo (90 mmol
kg–1 wet mass) is much larger than the amount of urea lost by
excretion (7 mmol kg–1 wet mass). Therefore, urea synthetic
rates are probably altered under dilute seawater exposure during the 8-month
stage, perhaps by changes in substrate flux through the pathway or the
availability of cofactors and/or modulators.

Higher urea levels in the older (8-month) embryos do not correlate with
higher OUC activities. As the embryo grows between 4 and 8 months
(approximately a 12-fold increase in embryonic tissue mass), there is probably
a corresponding increase (∼12-fold) in the amount of OUC enzymes to
maintain the per g wet mass (or per mg protein) activity. The urea tissue
content will also be influenced by the rate of urea loss to the environment.
One factor that plays a role in the rate of urea loss is the extent to which
embryos can exchange materials with the external seawater environment, that
is, the relative permeability of the egg case and the embryonic membranes. In
our study the egg case was removed, but it is not known to be a barrier to
urea and other small molecular mass solutes (for a review, see
Kormanik, 1995). The egg cases
of oviparous elasmobranchs develop an opening to the environment when the
albumin within the case disappears, approximately 1/3 of the way into
embryonic development (Koob,
1999). At 4 months post-oviposition, therefore, the embryos used
in the present study have only recently been exposed directly to seawater. In
the face of such a large tissue:water urea gradient and a comparatively low
rate of urea loss, it is very likely that the embryonic membranes are
relatively impermeable to urea, as reported for adult elasmobranchs (see
Introduction). Kormanik (1995)
points out that the large size of the skate embryos compared to teleost
embryos would also be advantageous in reducing urea loss, since a low surface
area:volume ratio would facilitate urea retention. Indeed, older embryos
(8-month) had significantly lower urea excretion rates (eightfold). Whether
the lower rates of urea loss are simply due to a lower surface area:volume
ratio in the older, larger embryos, or also to the development of urea
retention mechanisms by the gill and kidneys (see Introduction), is not
known.

CPSase III activity in both 4- and 8-month embryos was, not surprisingly,
much lower than activities in adult liver. Lechenault et al.
(1993) reported that the liver
constituted only 4.8–6.8% of the total body wet mass of Scyliorhinus
canicula (an oviparous dogfish) neonates. If one assumes that all of the
CPSase III activity is localized in the liver of R. erinacea embryos,
then we have severely underestimated the hepatic CPSase III activity in
embryos. This may not be the case, because in 8-month muscle tissue the CPSase
III activity was 0.008±0.001 μmol min–1
g–1 or 44% of the total whole embryo CPSase III activity
(Table 3). Thus, a significant
component of CPSase III activity resides in the skeletal muscle tissue, and
possibly other tissues as well. Skeletal muscle CPSase III has been reported
in teleost fish (Julsrud et al.,
1998; Kong et al.,
1998; Lindley et al.,
1999; Todgham et al.,
2001; Steele et al.,
2001), but to our knowledge never in elasmobranchs. In addition,
Gsase, OTCase and arginase activities in skeletal muscle tissue were 18%, 70%
and 13% compared to whole embryo enzyme activities, respectively
(Table 3). It appears,
therefore, that skeletal muscle may play a role in urea synthesis, at lease
during early developmental stages.

Activities of OTCase and arginase presented in the current study are much
higher (4- to 70-fold) than those reported by Read
(1968b) in R.
binoculata (OTCase ∼0.2 μmol min–1
g–1 wet mass; arginase ∼1.7 μmol
min–1 g–1 wet mass; skate mass ∼0.1 g).
This is especially surprising since Read performed his assays at a higher
temperature (38°C) compared to the present study (26°C). The
discrepancy may be due to species or methodological differences.

We predicted that if embryos were capable of acclimating to 75% SW, they
would decrease the rate of urea synthesis by lowering the levels of OUC enzyme
activities and/or increase the rate of urea excretion, in order to achieve a
lower tissue content of urea. There were several distinct differences in the
response of 4- and 8-month old embryos to 75% SW, but at both stages there
were no significant changes in OUC enzyme activities
(Table 3). However, flux
through the OUC may be modulated by the intracellular levels of substrates,
cofactors, modifiers and/or other cellular conditions. Goldstein and Forster
(1971) reported an increase in
renal urea clearance, but no change in total body urea clearance in adult
little skates exposed to 50% SW for 5 days. They suggested that urea synthesis
may be diminished in dilute seawater (although this has not been tested in
adults), which would explain the significantly lower tissue urea levels under
these conditions (e.g. Price and Creaser,
1967; Goldstein and Forster,
1971). Indeed, we have discovered a downregulation of hepatic
arginase in adult skates acclimated to 75% SW (S. Steele, P. Yancey, P.
Wright, manuscript submitted for publication).

After 5 days in dilute seawater, embryos were recovered in 100% SW for 3
days. An initial decrease in urea excretion rates might enhance the retention
of urea to bring levels close to pre-dilute seawater exposure. The results are
opposite to what we expected in that urea excretion rates increased
immediately and dramatically (11-fold) in the 8-month embryos
(Fig. 2) and marginally, but
significantly, in the 4-month embryos (Fig.
1). The explanation for this counter-intuitive response is
unknown. Such an increase could be related to a transient increase in water
efflux, as tissue water content would presumably recover towards control
levels. This would in turn increase excretion rates if, for example, urea
retention mechanisms were not equipped to retain urea under such conditions in
early life stages. Indeed, it is also interesting that recovery in 100% SW
(8-month embryos) was accompanied by a significant increase in ammonia
excretion rates (Fig. 3). In
adult little skates, ammonia excretion was unchanged by exposure to 50% SW or
recovery in 100% SW (Goldstein and Forster,
1971). These developmental differences may relate to a higher
proportion of nitrogenous wastes excreted as ammonia in embryos (up to 66%)
versus adults (19%; Goldstein and
Forster, 1971).

Ammonia excretion was detected in 8-month, but not 4-month, embryos. It is
not clear why the rate of ammonia excretion was below the level of detection
very early in development. Ammonia excretion constitutes approximately 19% of
total nitrogen excretion in the adult little skate
(Goldstein and Forster, 1971),
which is lower than values obtained for 8-month embryos in the present study
(up to 66%). Clearly, there are a number of ontogenic changes in nitrogen
metabolism that alter the proportion of nitrogen excretory products throughout
development.

In summary, ureosmotic mechanisms appear to be active very early in
development in the little skate, as indicated by the presence of significant
levels of OUC enzymes and the ability of these embryos to survive and recover
from dilute seawater exposure. The ratios of urea to compatible and
counteracting osmolytes (e.g. TMAO, other methylamines and amino acids) in
skate embryos are similar to those found in adult elasmobranchs. Upon exposure
to dilute seawater, 8-month old embryos downregulate tissue contents of these
osmolytes as observed in adult elasmobranchs; however, they are unique to
their adult counterparts in that urea excretion is increased in this milieu.
Finally, although independent ureosmotic regulation is in place as early as 4
months, significant developmental changes in nitrogen metabolism and
excretion, as well as osmoregulation, occur over the time period we examined
between 4 and 8 months post-conception.

Appendix

Example: 4-month urea excretion versus urea production at Time 0,
control (N=4)

Therefore, at this stage, the capacity of CPSase III to produce urea is 2.5
times that which is excreted.

ACKNOWLEDGEMENTS

The authors wish to thank Tammy Rodela for her careful management and
feeding of the little skate colony. This work was funded by a NSERC Discovery
grant to P.A.W., a Stanley Rall (Whitman College) grant to P.H.Y. and an
Ontario Graduate Scholarship to S.L.S.

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